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Preparation and Immunoaffinity Depletion of Fresh Frozen Tissue Homogenates for Mass Spectrometry-Based Proteomics in the Context of Drug Target/Biomarker Discovery

  1. Author:
    Prieto, DaRue A
    Chan, King
    Johann, Donald J
    Ye, Xiaoying
    Whitely, Gordon
    Blonder, Josip

  2. Author Address

    Cancer Research Technology Program, Protein Characterization Laboratory, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, PO Box B, Frederick, MD, 21702, USA., Wintrop P Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA., Cancer Research Technology Program, Antibody Characterization Laboratory, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, PO Box B, Frederick, MD, 21702, USA., Cancer Research Technology Program, Antibody Characterization Laboratory, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, PO Box B, Frederick, MD, 21702, USA. blonderj@mail.nih.gov.,
    1. Year: 2017
    2. Date: 2017
  2. Journal: Methods in Molecular Biology
    1. 1647
    2. Pages: 71-90
  4. Type of Article: Article
  5. Article Number: 71-90
  6. ISSN: 978-1-4939-7200-5
  1. Abstract:

    The discovery of novel drug targets and biomarkers via mass spectrometry (MS)-based proteomic analysis of clinical specimens has proven to be challenging. The wide dynamic range of protein concentration in clinical specimens and the high background/noise originating from highly abundant proteins in tissue homogenates and serum/plasma encompass two major analytical obstacles. Immunoaffinity depletion of highly abundant blood-derived proteins from serum/plasma is a well-established approach adopted by numerous researchers; however, the utilization of this technique for immunodepletion of tissue homogenates obtained from fresh frozen clinical specimens is lacking. We first developed immunoaffinity depletion of highly abundant blood-derived proteins from tissue homogenates, using renal cell carcinoma as a model disease, and followed this study by applying it to different tissue types. Tissue homogenate immunoaffinity depletion of highly abundant proteins may be equally important as is the recognized need for depletion of serum/plasma, enabling more sensitive MS-based discovery of novel drug targets, and/or clinical biomarkers from complex clinical samples. Provided is a detailed protocol designed to guide the researcher through the preparation and immunoaffinity depletion of fresh frozen tissue homogenates for two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS/MS)-based molecular profiling of tissue specimens in the context of drug target and/or biomarker discovery.

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External Sources

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  2. DOI: 10.1007/978-1-4939-7201-2_5
  3. PMID: 28808996
  4. View record in Web of Science®
  5. WOS: 000429733200006

Library Notes

  1. Fiscal Year: FY2016-2017
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